A. Field of the Invention
The present invention pertains to the engineering of foreign vertebrate gene constructs by recombinant DNA techniques for the more efficient processing and secretion of foreign genes in insect systems. Particularly, the present invention relates to replacing foreign vertebrate protein signal peptide sequences with protein signal sequences from insect cell secreted proteins.
B. Description of the Related Art
Baculovirus expression vectors (BEVs) have become extremely important tools for the expression of foreign genes, both for basic research and for the production of proteins with direct clinical applications in human and veterinary medicine (W. Doerfler, Curr. Top. Microbiol. Immunol., 131:51-68 (1968); V. A. Luckow and M. D. Summers, Bio/Technology, 6:47-55 (1988a); L. K. Miller, Annual Review of Microbiol., 42:177-199 (1988); M. D. Summers, Curr. Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1988)). BEVs are recombinant insect viruses in which the coding sequence for a chosen foreign gene has been inserted behind a baculovirus promoter in place of the viral gene, e.g., polyhedrin (G. E. Smith and M. D. Summers, U.S. Pat. No., 4,745,051, which is incorporated herein by reference).
Several advantages may be enjoyed when employing the BEV system. One of these advantages is the strong polyhedrin promoter which directs a high level of expression of the insert (protein of choice). The newly expressed protein accumulates in large amounts within these infected insect cells. Thus, as a result of the relative strength of the polyhedrin promoter, many different gene inserts can be expressed at very high levels.
Because the polyhedrin gene is a non-essential gene for productive viral infection, another advantage of baculovirus expression vectors is that the recombinants are viable, helper-independent viruses. Also, baculoviruses are noninfectious for vertebrates, and are therefore relatively safe genetic manipulation agents.
Thus, baculoviruses have gained popularity as expression vectors because of the advantages presented above. The BEV system is currently being employed in over 700 laboratories for the overexpression and production of many different gene products. To date, more than 50 different genes have now been expressed by employing this system (V. A. Luckow and M. D. Summers, Bio/Technology, 6:47-55 (1988)).
The success of engineering the polyhedrin promoter for the expression of foreign genes is merely a first step indicative of the potential to genetically alter baculoviruses for pest control. Presently there are no reports of a recombinant viral pesticide, but clearly the technology is available and the potential is excellent for inserting foreign genes, encoding for proteins or peptides, which could be highly disruptive to some aspect of insect behavior or metabolism. As mapping of the molecular organization and function of the baculovirus genome and the temporal regulation of these functions is determined, it should be possible to select AcMNPV promoters, in addition to that of polyhedrin, which would be more useful for expressing genes with potential pesticidal activities. To be successful, however, more knowledge is needed of the regulation of baculovirus gene expression, as well as a better understanding of the critical target sites in the insect for such products. (M. D. Summers and G. E. Smith, in: Genetically Altered Viruses and the Environment, 319-329, Cold Spring Harbor Laboratory Press (1985) (B. Fields et al. Eds.)).
Theoretically, a highly insect-specific toxin or bioregulator could be expressed under the temporal control of any identified baculovirus promoter which, when expressed, could rapidly debilitate the insect. There are numerous sites for such products within an insect which, if successfully targeted, would increase the pesticidal effectiveness of the baculovirus many-fold. It might be possible to engineer viruses in ways to improve persistence in the environment, to improve virulence, or to expand host range within an acceptable and limited spectrum. With respect to an expanded host range, there are no viral or host factors identified that would allow genetic engineering to proceed on an informed and predictable basis. Basic research on the molecular biology of baculovirus gene function and regulation and the properties of the host insect that play a role in host range and virulence will be essential to develop these potentials and applications. (M. D. Summers and G. E. Smith, in: Genetically Altered Viruses and the Environment, 319-329, Cold Spring Harbor Laboratory Press (1985) (B. Fields et al. Eds.)).
There are several families of virus which are insect pathogenic. Particularly useful viruses are from the family Baculoviridae which infect only arthropods, and particularly the Order Lepidoptera. Baculoviruses used for insect control are erratic and slow acting for causing mortality. It has been suggested that a baculovirus be genetically engineered in a way which would disrupt the endocrine balance of the target insect and, therefore, act as a viral pesticide. (G. E. Smith and M. D. Summers, U.S. Pat. No. 4,745,051).
The use of baculovirus vectors relies upon the host cells being derived from Lepidopteran insects. The proteins expressed by the BEVs are, therefore, synthesized, modified and transported in host cells derived from Lepidopteran insects. Most of the genes that have been inserted and produced in the baculovirus expression vector system have been derived from vertebrate species.
Other baculovirus genes in addition to the polyhedrin promoter may be employed to advantage in a baculovirus expression system. These include immediate-early (.alpha.), delayed-early (.beta.), late (.gamma.), or very late (.delta.), according to the phase of the viral infection during which they are expressed. The expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation. Thus, the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes. Some delayed-early gene products, in turn, induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes. One relatively well-defined component of this regulatory cascade is IE1, a preferred immediate-early gene of Autographa californica nuclear polyhedrosis virus (AcMNPV). IE1 is expressed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene (L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. Virol.. 61:2091-2099 (1987)), as well as late genes (L. A. Guarino and M. D. Summers, Virol., 162:444-451 (1988)).
Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes. The combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the HindIII-k fragment of the baculovirus genome. For example, the 39K promoter region is linked to the heterologous gene of interest and expression is further controlled by the presence of IE1. ((L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. Virol., 60:215-223 (1986); L. A. Guarino, M. A. Gonzalez and M. D. Summers, J. Virol., 60:224-229 (1986)).
Additionally, when a combination of immediate-early genes with a delayed-early gene promoter region is used, enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region. Such enhancer sequences are characterized, by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited. For example, the hr5 enhancer sequence is linked directly (in cis) to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA. (L. A. Guarino and M. D. Summers, J. Virol., 57:563-571 (1986a); J. Virol., 60:215-223 (1986b); L. A. Guarino, M. A. Gonzalez and M. D. Summers, J. Virol., 60:224-229 (1986)).
The polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. In addition to the polyhedrin gene, the p10 genes ("10K genes") of baculoviruses can also be utilized in BEVs. (J. M. Vlak et al., J. Gen. Virol., 69:765:76 (1988)).
Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers (U.S. Pat. No. 4,745,051) will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovirus infection progresses. Thus, gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins such as human tissue plasminogen activator. As a consequence, the expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.
At present, the only mode of achieving secretion of a foreign gene product in insect cells is by way of the foreign gene's native signal peptide. Because the foreign genes are usually from non-insect organisms, their signal sequences may be poorly recognized by insect cells, and hence, levels of expression may be suboptimal.
Heretofore, the efficiency of expression of foreign gene products seems to depend primarily on the characteristics of the foreign protein. On average, nuclear localized or non-structural proteins are most highly expressed, secreted proteins are intermediate, and integral membrane proteins are the least expressed. One factor generally affecting the efficiency of the production of foreign gene products in a heterologous host system is the presence of native signal sequences (also termed presequences, targeting signals, or leader sequences) associated with the foreign gene. The signal sequence is generally coded by a DNA sequence immediately following (5' to 3') the translation start site of the desired foreign gene.
The expression dependence on the type of signal sequence associated with a gene product can be represented by the following example: If a foreign gene is inserted at a site downstream from the translational start site of the baculovirus polyhedrin gene so as to produce a fusion protein (containing the N-terminus of the polyhedrin structural gene), the fused gene is highly expressed. But less expression is achieved when a foreign gene is inserted in a baculovirus expression vector immediately following the transcriptional start site and totally replacing the polyhedrin structural gene.
Insertions into the region -50 to -1 significantly alter (reduce) steady state transcription which, in turn, reduces translation of the foreign gene product. Use of the pVL941 vector optimizes transcription of foreign genes to the level of the polyhedrin gene transcription. Even though the transcription of a foreign gene may be optimal, optimal translation may vary because of several factors involving processing: signal peptide recognition, mRNA and ribosome binding, glycosylation, disulfide bond formation, sugar processing, oligomerization, for example.
The properties of the insect signal peptide are expected to be more optimal for the efficiency of the translation process in insect cells than those from vertebrate proteins. This phenomenon can generally be explained by the fact that proteins secreted from cells are synthesized as precursor molecules containing hydrophobic N-terminal signal peptides. The signal peptides direct transport of the select protein to its target membrane and are then cleaved by a peptidase on the membrane, such as the endoplasmic reticulum, when the protein passes through it.
While it has been recognized that the signal sequence associated with a foreign inserted mammalian gene is recognized by the insect cell system and cleaved at the correct sites, the present invention is premised on the use of an insect signal sequence instead of or together with the mammalian signal sequence so as to further enhance the expression of the foreign gene in the insect cell system.